Fungi have an illustrious record in drug discovery. The range of active compounds that have been identified include antibiotics, anti-cancer drugs, blood glucose regulators, cholesterol inhibitors, psychotropic drugs, immunosuppressants and even fungicides. Among the important modern fungi-based antibiotics are penicillin, cephalosporins, fusafungine, usnic acid, fusidic acid, fumagillin, brefeldin A, verrucarin A and alamethicin, Fungi are also the source of prominent immunosuppressants, including cyclosporin, mycophenolic acid and mizoribine. Certain statins are derived from fungi, including lovastatin, the pravastatin precursor mevastatin, and the simvastatin precursor monacolin J. And perhaps counterintuitively, fungi are also a source of antifungal drugs such as griseofulvin, echinocandins, strobilurin, azoxystrobin, caspofungin, and micafungin.
Medicinal species are perhaps more common than not among mushrooms. For instance, at least 650 species from 182 genera of hetero- and homo-basidiomycetes have been reported to contain polysaccharides that possess anti-tumor and/or immunostimulative properties; see S. V. Reshetnikov, S. P. Wasser, and K. K. Tan, “Higher Basidiomycetes as a source of antitumour and immunostimulating polysaccharides (Review),” Int. J. Med. Mushrooms, 3:361-394 (2001). These polysaccharides appear in fruit bodies, culture mycelia and culture brothers. However generally the fruit bodies are the richest source of the active medicinal principle and contain a greater diversity of bioactive polysaccharides.
Extractions of medicinal mushrooms fall into primarily two categories: hot water methods to isolate polysaccharides and glycoproteins, versus ethanol-based methods to isolate terpenoids, sterols, and other less polar compounds. These separations may be used in complementary ways, for instance to obtain both purified mycogenous carbohydrates and purified terpenoids. In some cases these extractions are accompanied by enzymatic treatments, particularly for breaking down polymeric bulk structures formed from chitin and/or chitosan.
Common examples of hot water extracts used to isolate bioactive carbohydrates and glycoproteins include those from Reishi (Ganoderma lucidum), Maitake (Grifola frondosa) and Shiitake (Lentinus edodes) mushrooms. The Reishi samples typically involve further purification with salts, alkali and dimethylsulfoxide solvent. The Maitake samples typically involve a deproteination step to free the conjugated polysaccharides. Shiitake extracts typically arise from a hot water extract of powdered mycelia, held for 50-60 h at 40-50° C. and allowed to undergo partial hydrolysis by endogenous enzymes, then the residue is extracted with 60° C. water, and the filtrate is freeze-dried to obtain “LEM” product; a different valuable fraction (“LAP”) is obtained by using a 1:4 water-in-ethanol solution for the filtrate.
The division between aqueous and solvent-based steps is evident in what Mizuno et al. (1999) have characterized as the typical approach to extract anti-cancer polysaccharides from mushroom fruit-bodies, mycelium and liquid media. First dried material (mushroom powder or mycelium) is repeatedly heated in 80% ethanol to extract and remove low molecular weight substances. The media for serial crude fractionations are water (100° C., 3 h, fraction 1), 1% ammonium oxalate (100° C., 6 h, fraction 11), and finally 5% NaOH (80° C., 6 h, fraction 111). The resulting polysaccharides are then further purified by ethanol, fractional precipitation, use of acetic acid to re-precipitate, ion-exchange chromatography, gel filtration chromatography and affinity chromatography. See T. Mizuno, “The extraction and development of antitumour-active polysaccharides from medicinal mushrooms in Japan,” Int. J. Med. Mushrooms, 1:9-29 (1999).
Purities by that method can exceed 99%, but a thriftier and much more efficient protocol achieves 87% purity (e.g., for the beta glucan lentinan from Leninus edodes) by simply precipitating the carbohydrate in ethanol and freeze-drying in liquid nitrogen, followed by lyophylization (evaporation), extraction with boiling water, and reiterative cycles of centrifuging and redissolving in 95% ethanol. See A.-T. Yap and M.-L. M. Ng, “An improve method for the isolation of lentinan from the edible and medicinal shiitake mushroom, Lentinus edodes (Berk.) Sing. (Agaricomycetdeae)” Int. J. Med. Mushrooms, 3:6-19 (2001).
Where the compound of interest is an exudate, the methods are even easier: e.g., nearly neat ethanol is used to absorb the compound, then a precipitate is collected by centrifugation, then the precipitate is redissolved in and for two days dialyzed against distilled water, and analysed by gel filtration chromatography (Babitskaya et al., 2000).
Extraction of less polar organics proceeds by other methods. Typically these involve organic solvents, in particular ethyl acetate, methanol or ethanol, though others are also used. See, e.g., I. J. Nieto and C. A. Carolina, “Triterpenoids and fatty acids identified in the edible mushroom Pleurotus sajor-cajú,” J. Chil. Chem. Soc., 53(2):1515-1517 (2008). An example of recent innovation in this area is the use of electrophoretic methods for inexpensive extraction of triterpenoids from Reishi. See C.-R. Cheng, “Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone refining,” Food Chem., 130(4):1010-1016 (2012).
What is common for all of these methods is the objective of enhancing the potency and safety of fungal compounds for medicinal use. The emphasis on purification, isolation and identification has arisen because in most cases formulation designers do not know how to optimize the identities, ratios or concentrations of fungal molecular natural products either during fungiculture or during harvesting and extraction. In fact, often the physiological basis of the medicinal effects in humans is also not well understood. Thus there is an ongoing need for improved extraction methods and improved medicinal formulations of fungal species.